The myelin sheath forms by the spiral wrapping of a glial membrane around an axon. The;mechanisms responsible for this process are unknown but are likely to involve changes in the glial cell cytoskeleton. Recent data from our laboratory strongly suggest that regulation of actomyosin assembly via phosphorylation of myosin light chain (MLC) is an important aspect of this process. We have found that Rho-associated kinase (ROCK) and myosin light chain kinase (MLCK), two of the major kinases involved in MLC phosphorylation, have very distinct roles on myelination. In Schwann cell-neuron cocultures, inhibition of ROCK results in atypical Schwann cell morphology, with cells that branch aberrantly and form multiple, small independent myelin segments along the length of axons, each with associated nodes and paranodes. This organization partially resembles myelin formed by oligodendrocytes. By contrast, inhibition of MLCK appears to prevent myelination by interfering with the spiral wrapping of the myelin sheath around the axon. We have also found that MLC phosphorylation is robustly, but transiently, activated at the onset of myelination during development. In this study we will test the hypothesis that ROCK and MLCK, two of the major pathways regulating MLC phosphorylation and hence actomyosin activity, are directly involved in the control of myelin morphology and its wrapping around the axon. The overall goal of this project is to provide novel insights into the mechanisms that regulate myelin morphology and formation in the PNS and CNS. A basic understanding of the molecular machinery of myelination should aid in the development of new therapeutic strategies to promote remyelination in diseases;such as multiple sclerosis and peripheral demyelinating neuropathies.